Coastal Coccolithophores (Guest Ed. R.W. Jordan)
J. Nannoplankton Res., Special Issue, 4, 2019, pp. 55–61 © 2019 International Nannoplankton Association
ISSN 1210-8049 Printed by The Sheridan Press, USA

55

Using mussels to study coastal calcareous nannoplankton
associations: Case study from the west coast of Portugal
Gonçalo A. Prista*

Instituto Dom Luiz, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal & Sociedade de História Natural, Travessa Florêncio
Augusto Chagas nº8, R/C Apartado 25-2564-909, Torres Vedras, Portugal; *gaprista@fc.ul.pt

Mário A. Cachão

Instituto Dom Luiz, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
Manuscript received 9th March, 2016; revised manuscript accepted 22nd August, 2016

Abstract Coccolithophore studies usually involve cruises and the filtration of large amounts of water. With theobjective of reducing sampling costs and simplifying the collecting method, we tested Mytilus galloprovincialis, acommon, local, rocky-shore filtering bivalve, as a biogenic coccolithophore sampler. A small device, named a musseldrome, was developed to harvest the mussels’ abundant pellet filaments. Mussel pellets were collected every week,
for seven weeks, between May and July of 2014. Abundant coccoliths were found, being dominated by Gephyrocapsa
oceanica, followed by Coccolithus pelagicus and Coronosphaera mediterranea. Coccospheres were also present,
dominated by G. oceanica, frequently present as agglomerates. The results show that the filtering process undertaken
by mussels has little effect on coccoliths, coccospheres or even cell clusters. Preliminary results show that mussels
can be used as samplers for calcareous nannoplankton community structure studies, since the estimated concentrationof each species found were in agreement with results from water-column coccolithophore concentrations from offwestern Portugal. The advantage of this method over other sampling techniques is that weekly averages of plankton
concentrations can be obtained – something that the usual devices used in sampling (phyto)plankton cannot duplicate.

Keywords Coccolithophores, coastal assemblages, mussels, calcareous nannoplankton, sampling methods
1. Introduction
Coccolithophores constitute a major component of planktonic communities throughout the world’s oceans (Okada & McIntyre, 1977). They are of remarkable interest,
being among the main primary producers, and playing
a distinct role in oceanic ecosystems (Field et al., 1998;
Balch, 2004). They have a significant role in the global
carbon and sulphur cycles (and therefore climate regulation) through direct involvement in ocean–atmosphere gas
exchange (Malin & Steinke, 2004), which arises from thefact that they are among the most important pelagic calcifying organisms in the modern ocean (Baumann et al.,2005), accounting for up to 20% of total carbon fixation
in some systems (Poulton et al., 2007). For these reasons,studies on coccolithophores have gained importance overthe past decade; however, studying modern communities
is usually expensive, since boat trips are necessary forsampling. Moreover, coccolithophores are mainly oceanic, and their role in coastal environments is still poorly
understood (Ferreira & Cachão, 2005), although somespecies are known to occur in neritic environments when
the conditions are right, such as Gephyrocapsa oceanica(Ferreira & Cachão, 2005), or have even become specialised in such environments, such as Coccolithus pelagicus(Cachão & Moita, 2000).
With the objective of reducing costs, simplifying thesampling method, and increasing our knowledge regarding coccolithophores in coastal environments, filtering
species of coastal bivalves were examined for their role ascoccolithophore samplers.
Mussels appear to be the most promising organisms forsuch an approach, since they filter large quantities of water
per day (up to ~30L; Riisgård et al., 2011), and have a particle size selection process (Bougrier et al., 1997; Norén etal., 1999; Riisgård et al., 2011), in which particles >4μm
go through a non-selective process (Møhlenberg & Riisgård, 1978), separating suspended particles from algae,
the former being expelled with the mucus (Riisgård et al.,2011). Mussels produce two kinds of faeces – intestinaland glandular. When ample food is available, the intestinal
faeces constitute the vast majority of the excreted material,but, when little food is present, this decreases considerablyin amount. The intestinal faeces consist of less-digested
material that is transported directly into the hindgut, bypassing the digestive diverticula. The glandular faeces
stem from food that gets into the digestive diverticula,and so is much more thoroughly digested (Riisgard et al.,2011). Both expelled faeces types are deposited beneath56

Prista, Cachão

the mussel beds, and contain undigested and rejected material.
A small structure – the musseldrome (Fig. 1) – wasdeveloped to collect mussel pellets from a selected set ofmussel individuals. The mussel faecal pellets were thenprepared and sampled for the identification and countingof calcareous nannoplankton, in the form of (cocco)lithsand (cocco)spheres. Coccoliths of calcareous nannoplankton were found to be abundant among the suspended particles, and present a useful proxy for modern community
structure studies. Coccospheres were also present, andthey provide an indication of the present community structure. Our primary results show that mussels have great potential for use in coccolithophore studies.
2. Material and methods
The study was performed at Peniche (39˚N, 9˚W), on thewestern coast of Portugal, using individuals of the species
Mytilus galloprovincialis.
The musseldrome consists of an adapted, inverted 5L
plastic carboy. First the carboy was cut in half. A net witha 0.5cm-diameter mesh was sewn in place, functioning
as a barrier between each half of the carboy and, at thesame time, unifying the two halves. The capped end of thebottle was used as the pellet collection end; the other half
was cut, creating slits for seawater to circulate through.
The structure was placed in the water-column, 4.5m above
the seafloor, so that the musseldrome was never out of thewater (at the low tide of a high-amplitude tide, it stood
1.5m below the water surface). To stabilise it, a 150g fish-

Figure 1: Schematic of the musseldrome. A 5L carboy cut in half, witha net placed between the two halves. The upper and lower parts of the
carboy are sewn together with a saltwater-resistant twine to close themusseldrome and keep the mussels inside. Slits are made in the upper
part to allow water to flow through and feed the mussels. A fishing weightis tied to the cap to keep the musseldrome fixed in position

ing weight was tied to the cap. Six specimens, with initial
sizes of between 27 and 47mm, were introduced throughthe slits in the upper half.
Although removed from a nearby mussel bed, the six
individuals were left inside the musseldrome for onemonth prior to the first faecal filament collection, whichwere then collected weekly for seven sequential weeks.
Pellet collection was performed by removing the musseldrome from the water, and carefully opening the cap of
the collection bottle, so as not to lose any of the sample.
After decanting the sample into a bottle, the musseldrome
was closed and placed back in the water.
Samples were prepared for observation according to
the following four-step method: 1) volume makeup andsubsampling; 2) decantation; 3) drying; 4) preparation for
microscopy screening. The first step was performed insitu. Filtered water was added to the bottle the pellets were
decanted into to make up the volume to 2L. This pellet suspension was homogenised, and three subsamples of 50ml
each were prepared. The second step was performed in thelab. Each subsample was subjected to ultrasonic cleaningfor 15 minutes (to disaggregate the organic matter) in a
P Selecta ultrasonicator, and was then homogenised, after
which 1ml was added to buffered water and left to settle ina Petri dish over a coverslip (random settling; Flores & Sierro, 1997). After 24 hours, the preparations were dried at
60˚C. Finally, each sample was prepared for microscopic
observation. The dried coverslip was removed from thePetri dish and mounted on a slide using a synthetic resin
medium (EntellanTM), heated to remove air-bubbles, andleft overnight to solidify.
The pellet contents were observed under a Zeiss Ortholux II Pol-BK transmitted-light binocular microscope,using polarised light and at a magnification of 1250x.
To calculate the final concentration of liths and spheres
per litre, we used the equations developed by Riisgård etal. (2014) for Mytilus edulis, since there are no specificequations yet for M. galloprovincialis. The equations are
dependent on mussel size, which changed due to growthover the seven-week sampling period (see Table 1). We assumed a linear growth over this period, so as to adjust theequations for calculation of the final concentration. The
equation used to calculate the amount of litres per hour
filtered by each mussel was:F = 0.00135L2.088 Lh-1
, where L is the mussel length. This equation was devel-

Coastal nannoplankton associations of Portugal

oped for mussels at 12˚C. The same authors estimated anincrease of 2.5% in filtration rate for each 1˚C rise in temperature. We adjusted our results to reflect a mean watertemperature of 18˚C. The final results can be seen in Table
2.Table I
Mussels
M#1
M#2
M#3
M#4
M#5

M#6

Length (mm) Length (mm) Difference
25/04/2014

26/07/2014

(mm)

34

58

24

32
47
43
27
28

54
64
60
49
51

22
17
17
22
23

Growth
(%)

68.75
36.17
39.53
81.48
82.14

70.59

Table 1: Mussel size variation over the one-month acclimatisation andseven-week sampling period

3. Results
The data correspond not to discrete samples, but to weekly-averaged samples. Some variation was observed overthe seven-week period (see Table 3); however the coccolith species distribution remained constant, with a dominance of G. oceanica, followed by G. muellerae withCoronosphaera mediterranea, Helicosphaera carteri andC. pelagicus as the abundant species. The remaining species were low in abundance/presence, with four species
comprising 2% of the coccoliths counted.
Gephyrocapsa oceanica coccospheres were particularly abundant in the mussel pellets, in the presence of agglomerates and G. muellerae. Other species showed scarceabundances, usually being absent (see Table 4).
4. Discussion and conclusions
The more surprising results were the presence of coccospheres. Not only were several coccospheres present in themussel pellets, but, in the case of G. oceanica, they were
found frequently in the agglomerates (see Figure 2). Also,
coccospheres of C. mediterranea, a non-placolith-bearing
species, were present on the slides. This means that neither
the filtering process undertaken by the mussels, nor theultrasonication step in the sample preparation process, affected the coccospheres.
For comparison of our results with those of otherstudies on the present-day community structure of coccolithophores along the west coast of Portugal, we used the
Guerreiro et al. (2015) study from the Nazaré Canyon andadjacent western continental margins and slopes, whichused seafloor sediment surface samples. A close agreement of our results with those of Guerreiro et al. (2015)57

was found. Calcidiscus leptoporus, C. mediterranea andC. pelagicus showed similar occurrences to those from theNazaré Canyon. Gephyrocapsa oceanica and H. carteri
showed results similar to the ones from the Extremadura
Slope. This could mean that Peniche is under the influenceof both oceanographic realms, since it is between the Extremadura shelf slope and the Nazaré Canyon (see Figure
3).
Gephyrocapsa muellerae, with a mean relative abundance of 10%, was the only species found not to agreewith the results found in Guerreiro et al. (2015), whichwere 25% from the Extremadura Slope and 40% at theNazaré Canyon.
The most probable explanation for this discrepancy
regarding G. muellerae is the fact that mussel pellets arevery rich in organic matter, making it difficult to observe
smaller coccolithophore species (see Figure 4). This could
explain the lower values for this particular – smaller – species. For this reason, only Emiliania huxleyi and Gephyrocapsa ericsonii coccospheres were studied.
The other species present in our study were not taken
into account in the Guerreiro et al. (2015) analysis, so
there is no data available for comparison; however, thespecies for which we have no data represent less than 2%of our total abundances.
Our preliminary results showed a reasonably good
match, in terms of the general structure and proportionsof the coccolithophore community, between the data obtained by musseldrome and that from traditional phytoplankton sampling methods.
A study by Moita et al. (2010) found a strong presenceof H. carteri and C. mediterranea in nearshore and coastal
samples along the Peniche coast during the summer. This
is in agreement with our results, which identified these two
species as two of the most relevant in the coccolithophore
assemblages. Ferreira & Cachão (2005) showed that G.
oceanica increased in abundance towards the shore on the
Guadiana, Algarve coast, while G. muellerae showed the
opposite behaviour. This is in accordance with our results,
with their strong dominance of G. oceanica, and may explain the low presence of G. muellerae in our study.
The results from using mussel pellets for coastal calcareous nannoplankton studies are promising. The use ofmussels also provides data representing nearshore coccolithophore communities, for which, at present, there arefew data. Now that the methodology has been proven to58
Table II
Week
5
6
7
8
9
10
11

12

Prista, Cachão

M#1

577.35639.27704.48773.00844.84920.01998.52Filtration rate per mussel with size variation (L/week at 18˚C)

1080.39

M#2

1047.24
1111.95
1178.71247.47
1318.28
1391.121466.021540.44

M#3

889.54949.061010.58
1074.131139.71207.29
1276.92

1346.23

M#4

436.87490.62547.63607.90671.46738.33808.50882.00M#5

473.07531.62593.75659.46728.77801.69878.25958.84

M#6

TOTAL (L)1254.237062.14662.37734.91811.39891.80976.201064.57
1156.95

4086.444457.434846.525253.765679.246123.016585.15

Table 2: Filtration rate per mussel (L/week), with size variation, at 18˚C. The week number starts at five because the first four weeks correspond to theacclimatisation period. Although this period is not applicable to our results, it is important because mussel growth occurred during this time

Table 3: Lith concentration/L for each sample and species. Go – G. oceanica, Cp – C. pelagicus, Gm – G. muellerae, Hc – H. carteri, Sp – S. pulchra, Bb – B. bigelowi, Coron – C. mediterranea, Scy sp. – Scyphosphaera sp., Cl – C. leptoporus

Table 4: Mean values of coccosphere concentration/L for each week. Go – G. oceanica, Gm – G. muellerae, Ge – G. ericsonii, Cp – C. pelagicus,
Bb – B. bigelowii, Eh – Emiliania huxleyi, Cl – C. leptoporus, Coron – C. mediterranea

produce results, new campaigns are being prepared togather further data using mussels as coccolithophore samplers. Future works will aim at comparing mussel pellet
data with water-column data collected from the vicintiy ofthe musseldrome. Such a study will be carried out throughout the year, so that the procedure can be validated/understood for different seasons. This is particularly important,since the Portuguese west coast presents seasonal upwelling, which induces changes in phytoplankton communities. The morphological details of the coccoliths will also

be addressed in upcoming sampling campaigns.
Finally, the several processes by which particles arriveat the mussel colony, and that affect the mussels’ uptake ofcoccolithophores, are still open questions in such a study.
The future development of this project will aim to understand those processes and how they impact this method ofnearshore coccolithophore community study.
Acknowledgements
This work was supported by the Portuguese Scientific

Coastal nannoplankton associations of Portugal

59

Figure 2: Top row, left to right – Braarudosphaera bigelowii coccosphere, Calcidiscus leptoporus coccosphere, Coronosphaera mediterranea coccosphere. Middle row, left to right – Gephyrocapsa muellerae coccosphere, Coccolithus pelagicus coccosphere. Bottom row, left to right – Emiliania
huxleyi coccosphere, Gephyrocapsa ericsonii coccosphere, Gephyrocapsa oceanica eight-coccosphere agglomerate

Foundation (Fundação para a Ciência e Tecnologia), with
PhD grant SFRH/BD/95593/2013 attributed to GAP. The
authors would also like to thank fisherman José Maria Ricardo (Zezito) and his colleagues for giving us access to,and letting us use, their dock at the Peniche harbour. We
thank Dr Elisa Malinverno and an anonymous reviewer
for their comments.

Baumann, K., Andruleit, H., Bockel, B., Geisen, M. & Kinkel, H.

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